Silver Nanoparticles as an Intracanal Medicament: A Scoping Review

Silver nanoparticles (AgNPs) release Ag ions with potent bactericidal and anti-inflammatory effects. They have shown promising results as an intracanal medicament for removing Enterococcus faecalis (E. faecalis), a resistant bacterium associated with root canal failures. This review summarizes the role of AgNPs as an intracanal medicament. Original research articles on AgNPs as an intracanal medicament were searched in databases such as MEDLINE (PubMed), Scopus, and Embase, resulting in 24 studies. They showed that AgNPs effectively eliminated E. faecalis and reduced postoperative pain following root canal therapy. However, these effects should be further verified through clinical trials as most of the studies were in vitro.


Introduction
Root canal disease, a bioflm-mediated infection, is successfully managed through the reduction or elimination of the bioflm by adequate debridement, disinfection, and obturation with an excellent apical seal [1].
Resistant periradicular lesions are predominantly associated with Enterococcus faecalis (E.faecalis) and are often detected in the monocultures of the root canals of teeth [2].It is a facultative anaerobic Gram-positive microorganism strongly associated with secondary endodontic infections and failures.In addition, Fusobacterium nucleatum (F.nucleatum) is frequently associated with immature necrotic teeth [3], and Candida albicans (C.albicans) has been extracted from infected root canals along with E. faecalis [4].
E. faecalis can withstand extreme conditions due to its characteristic endurance to antibiotics, irrigant solutions, intracanal medicaments, and high alkaline pH [5].Besides, it forms bioflms that penetrate the dentinal tubules, complicating its elimination from root canals [6].Te biomechanical preparation and shaping of the root canal efciently reduce the number of microorganisms in the root canals but cannot remove them from the isthmuses, lateral canals, and apical deltas [7].
Calcium hydroxide (Ca(OH) 2 ) is a widely applied intracanal medicament between appointments to reduce canal bacteria.It is antibacterial, and during apexogenesis, or pulp capping, it stimulates hard tissue formation, dissolves the tissues, and promotes apical exudate elimination [8].It produces nonspecifc bactericidal action due to high alkalinity, as most microorganisms are eliminated at a pH of 9.5-12, and only a few survive at a pH of 11 or greater [9].Despite these antimicrobial properties, Ca(OH) 2 is inefective against polymicrobial infections or bacterial bioflms.However, its combination with antimicrobials such as chlorhexidine digluconate (CHX) has shown synergistic antibacterial action [10].
Lately, nanotechnology in dentistry has promoted the development of excellent biomaterials with unique physical, chemical, and biological properties [11].Te nanoparticles (NPs) have superior antibacterial efects due to their higher volume-to-surface area ratio and lesser particle dimension, which results in higher efective contact and larger reaction surface.Subsequently, they penetrate the dentinal tubules and produce a prolonged antibacterial impact at the infection site at reduced doses [12].
Among the various NPs, silver nanoparticles (AgNPs) are broad-spectrum and biocompatible, with sizes ranging from 1 to 100 nm [13,14].Tey release Ag ions, producing robust bactericidal efects against Gram-positive, Gramnegative, and multidrug-resistant bacteria [15,16].Although Ag is an inert material in the bulk state, it gets ionized by moisture and converts into a highly reactive state [17].AgNPs exert bactericidal action by destroying the cell envelope, inhibiting metabolic enzymes, and generating reactive oxygen species (ROS) [18].Moreover, they gather in the pits on the cell surface and denature the cell membrane leading to cell lysis and death.AgNPs are used as an intracanal medicament with an appropriate vehicle.Studies show their increased efectiveness compared against Ca(OH) 2 alone in removing E. faecalis [1,10], while a few evaluated their efects against other agents [4,19].
With this background, the present review summarizes the role of AgNPs as an intracanal medicament.

Inclusion and Exclusion
Criteria.Original research fulltext articles in English on the application of AgNPs as an intracanal medicament were included.Conference proceedings, recommendations, expert statements, technical reports, reviews, case reports, and nonoriginal papers were excluded.

Risk of Bias Assessment.
Te ROB-2 and Quin tools were used to assess the risk of bias in randomized controlled clinical trials (RCTs) and in vitro studies, respectively [37,38].Te ROB-2 tool has six criteria, including selection, performance, detection, attrition, and reporting bias, and showed a low risk of bias in both the included RCTs.Te Quin tool applies 12 criteria, of which only nine were considered relevant for the included studies.Tey were statements on aims and objectives, sample size calculation, sampling technique explanation, comparison group details, details on randomization, outcome measurements, statistical analysis, and presentation of results.Te criteria were scored, and the study was graded as high, medium, or low risk (Table 1).Tere was a low-to-medium risk of bias in the included in vitro studies.Two authors (SG and RA) did the risk of bias assessment, and disagreements were resolved by discussion among all the authors.

Data Extraction.
Te aims and objectives of the study, the type of study, AgNPs' form and concentration, the antimicrobial efect, results, and conclusions were recorded (Table 2).

Antimicrobial Efcacy.
Te positively charged AgNPs, with a large surface area, interact with the negatively charged cell membrane and exert an antimicrobial efect.Tey interact with the bacterial membrane's "building elements" leading to changes in structure and degradation and, fnally, cell lysis [39].Tey get internalized and generate ROS, which damage intracellular macromolecules such as DNA and proteins.

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Te Scientifc World Journal When the antimicrobial efect of various concentrations of AgNPs was evaluated, it was found that concentrations superior or equal to 300 μg/mL decreased E. faecalis growth by more than 90%, i.e., it was the minimum inhibitory concentration (MIC) for the bacteria.However, no growth was observed from 900 μg/mL onward, indicating that it was the minimum bactericidal concentration with the bacteriostatic efect at 300 μg/mL [27].Moreover, their antimicrobial efects at 300 μg/ml and 500 μg/ml were equivalent to the action of Ca(OH) 2 [27].Likewise, other concentrations of AgNPs ranged between 0.12 and 17 μg/ml of which 1.05 μg/ml was the MIC for E. faecalis [19].Besides, the AgNPs of aloe vera inhibited E. faecalis, S. mutans, and C. albicans in the concentration order 2000 μg/ml > 1000 μg/ml > 500 μg/ml [23].When various concentrations of AgNPs (0.03%, 0.04%, and 0.06%) were evaluated for their efcacy against F. nucleatum, they reduced the bacterial count in the increasing order of their concentration, with 0.04% of AgNPs being the lowest concentration efective against F. nucleatum [3].
However, a study showed the most signifcant reduction in the bacterial count with Ca(OH) 2 alone, followed by AgNPs combination with Ca(OH) 2 (1 : 2) and AgNPs alone during the frst and second weeks of exposure to the intracanal medicament [9].Moreover, another study showed that 2% CHX was most efective against E. faecalis and C. albicans, followed by AgNPs' combination with either 2% CHX and Ca(OH) 2 or AgNPs alone [4].

Other Efects.
AgNPs produced a more signifcant reduction of initial postoperative pain than Ca(OH) 2 alone, as indicated in the clinical trials on subjects undergoing endodontic treatment [24,30].Even though the Ag ions may cause gray-black discoloration of a crown over time, the AgNPs combined with Ca(OH) 2 caused no substantial tooth discoloration at one-week, one-month, and three-month intervals [20].Similarly, a comparison of crown discoloration after applying AgNPs and Ca(OH) 2 or AgNPs and graphene oxide showed no signifcant discoloration at 15 days.However, it was noted at the one-month interval [36].
3.6.Recent Advances.Some studies utilized mesoporous calcium-silicate NPs loaded with low-dose Ag ions, and Triton X-100 was used for the controlled delivery of AgNPs (M-AgTx).Tese particles inactivated a 28-day E. faecalis bioflm [33].Likewise, M-AgTx showed excellent antibacterial ability against E. faecalis and high substantivity on dentin [34].AgNPs with graphene oxide had an almost negligible efect on the root dentin microhardness compared to Ca(OH) 2 alone or when combined with AgNPs [35].

Discussion
Te included studies show that AgNPs as intracanal medicaments have potent antibacterial efects against E. faecalis, F. nucleatum, and C. albicans, comparable to Ca(OH) 2 and CHX.
A few studies revealed that green biosynthesis using plants and fungi developed AgNPs that were equally efective against E. faecalis [12,31,32], reducing costs and the risk of toxic byproducts.AgNPs biosynthesized from fungi showed antimicrobial efcacy similar to 2% CHX.Fungi require simple nutrients for growth, a cost-efective and easy process involved in the synthesis of AgNPs [12].Tey are like "nanofactories," producing particles with good monodispersity, size, and chemical composition.Likewise, plants such as A. paniculata and O. sanctum Linn were also applied for their synthesis [31].Te active compound in A. paniculata is andrographolide, which inhibits quorumsensing and microbial virulence factors.At the same time, O. sanctum Linn produces camphor, eucalyptol, eugenol, alpha-bisabolene, beta-bisabolene, and beta-caryophyllene responsible for its antimicrobial action.Tey were efective against S. aureus, C. albicans, and E. faecalis [31].Tese plants contain crude metabolites, including phenolic acid, favonoids, alkaloids, and terpenoids, which act as reducing agents to generate AgNPs from silver nitrate solution.No reports show whether biogenic NPs synthesized from various sources, including fungi or plants, are superior or inferior in their activities.However, those derived from fungi may be benefcial in manufacturing owing to the more signifcant amounts of metabolites produced.Besides, fungi produce antibiotics which act in synergy with the NPs [40].
AgNPs, when combined with Ca(OH) 2 , reduced the colony-forming units (CFUs) of E. faecalis within one week of the application when compared to Ca(OH) 2 alone [1,10,22].It was suggested that adding AgNPs to Ca(OH) 2 reduced its MIC against E. faecalis and increased the antimicrobial efcacy of the medicament [41].Moreover, AgNPs in gel form were used as a vehicle for Ca(OH) 2 , which ensured a prolonged interaction with bacterial cell walls, disrupted bacterial integrity, and reduced the CFUs of E. faecalis [42].Besides Ca(OH) 2 and CHX, triple and dual  [21] (i) Zero colony-forming units (CFUs) with nanocare with AgNPs Javidi et al. [22] (ii) Te CFUs observed after Ca(OH) 2 with AgNPs dressing were signifcantly less than those observed with Ca(OH) 2 alone Afkhami et al. [1] (iii) Reduced E. faecalis colonies in AgNPs with Ca(OH) 2 group Venkateshbabu et al. [19] (iv) Efective dentin disinfection from E. faecalis with AgNPs Saad and Alabdulmohsen [9] (v) Antibacterial efect of AgNPs was lower than that of Ca(OH) 2 or the combination of both materials Yadav et al. [4] (vi) 2% chlorhexidine was more efective as an intracanal medicament when compared to AgNPs and combination of AgNPs with Ca(OH) 2 or chlorhexidine against E. faecalis Haripriya and Ajitha [23] (vii) Ag-NPs of aloe vera had antimicrobial efect El Abbasy et al. [24] (viii) Reduced postoperative pain in the AgNPs group compared to the Ca(OH) 2 group Heidar et al. [25] ( Te Scientifc World Journal antibiotic pastes were compared with AgNPs [10].CHX (2%) has substantivity properties and an antimicrobial efect due to its positively charged molecules, which are adsorbed into the dentin and persist there for an extended duration, which prevents bacterial colonization.Te triple and dual antibiotic pastes showed more signifcant inhibition of E. faecalis but are associated with bacterial resistance, tooth discoloration, and changes in the dentin, leading to increased demineralization and fracture [10].However, AgNPs have a reduced risk of bacterial resistance due to the following four well-defned antimicrobial mechanisms [18,27]: frstly, they adhere to the cell wall and membrane surface; secondly, they penetrate the cell and damage intracellular structures such as mitochondria, vacuoles, ribosomes, and biomolecules, including proteins, lipids, and DNA; thirdly, they induce cellular toxicity and oxidative stress by generating ROS and free radicals; and fourthly, they modulate signal transduction pathways.Besides, they also modulate the immune system of the human cells and infuence the infammatory response, further inhibiting microorganisms [18].Tese modes of action reduce the postoperative pain for up to 24 hours following root canal treatment [30].
Although AgNPs have less probability of developing bacterial resistance, lately, some studies have reported that certain endodontic bacteria may develop resistance to them by various mechanisms [43].Tey include intrinsic (such as efux pumps, porins' downregulation, and chromosomal resistance genes) or extrinsic as point and adaptive mutations and plasmids with resistance genes) adaptation systems [43].AgNPs activate the envelope stress response, including positive charges into the bacterial cell wall.Subsequently, the bacteria equalize the electrical charge with the surface of AgNPs, leading to repulsion.Moreover, AgNP concentrations below the MIC lead to oxidative stress and the appearance of persistent bacteria such as E. faecalis.Tey develop cell dormancy, wherein the antibiotic binds to the bacteria but cannot kill them due to the downstream pathways' inactivation.Te nonlethal concentrations of AgNPs increase bacterial mutation due to ROS production and DNA damage.Tese mutations upregulate efux pump genes and downregulate porins, causing a resistant phenotype in bacteria.Te extrachromosomal genetic elements like plasmids enter the bacterial cells through active and passive mechanisms and promote lateral transfer of genes resistant to AgNPs.
Moreover, increased AgNP usage may stimulate coresistance and coregulation to metals and antibiotics in bacteria.Terefore, they would develop antibiotic resistance when exposed to nonbactericidal concentrations of AgNPs.Lastly, the sublethal exposure to AgNPs may enhance bioflm development, upregulate lipopolysaccharide formation, gene transfer, and efux pump genes, enhance the protein and sugar levels in the bioflm, and promote the development of more efective resistance mechanisms against the antimicrobial efect of AgNPs [43].
However, recent advances in AgNPs, like M-AgTx, may overcome these bacterial resistance mechanisms by coreleasing Ag + and TX-100, which eliminate bacteria in a concentration-and time-dependent manner [33].TX-100 increased the cell permeability and promoted the penetration of M-AgTx into the bacterial cell leading to cell perforation and breakdown.Tis mechanism of TX-100 decreased the alkaline and Ag + resistance of E. faecalis and enhanced the antibacterial efect of silver and hydroxide ions of M-AgTx against E. faecalis.Furthermore, combinations of graphene oxide with AgNPs improved the microhardness of treated dentine due to AgNPs' deposition inside the dentinal tubules [35].A study showed tooth discoloration with AgNPs, after their application as an intracanal medicament for a month [36].Usually, imidazolium-coated AgNPs produce staining like blood [44].Te discoloration is mainly due to dentinal tubule infltration.Terefore, their application should be limited to the root canal and not extend to the pulp chamber.Any medicament remains should be eliminated before the crown placement [44,45].
Te biodistribution and toxicity studies in rats and mice showed that AgNPs administered by inhalation, ingestion, or intravenous or intraperitoneal injections are found in blood and are toxic to the lungs, liver, kidneys, intestines, and brain [46].
Te toxicity is positively associated with free silver ion levels and is related to their nanosize, interrupting the bioactive molecules, eukaryotic cells, and tissues [45].Teir physical and chemical structures afect the cell response.Te oxidative stress of AgNPs toxicity produces free radicals, accumulating in the cell nucleus and cytoplasm.Due to their large surface area, AgNPs produce signifcant early toxicity, which decreases with time with their interaction with the organic compounds.
Moreover, they accumulate in the liver and spleen when used in high doses.Tey can also penetrate the blood-brain barrier by transsynaptic transport and accumulate in the brain [45,47].Nevertheless, any levels of accumulated silver are cleared from the body by eight weeks, although disproportionately high levels of AgNPs destroy the mitochondrial function.When applied in concentrations greater than 200 mg/kg body weight, they generate free radicals, release ROS, and cause cell damage.However, various animal models have not shown the toxic efects of AgNPs with an average size of 5 nm and concentration of 25 g/mL or when administered orally [45].A commercially available AgNPs colloid did not alter metabolism, blood, urine, vital signs, or physical or radiographic fndings and was excreted in feces, with only a minuscule amount absorbed [45].However, AgNP toxicology research on susceptible individuals and human exposure is understudied.Tere is a lack of detailed research on the efects of AgNPs on humans via various routes of exposure.Few studies evaluated whether AgNPs penetrated the physically and functionally intact human skin [48,49].Tey demonstrated that AgNPs (10-40 nm) penetrated the normal skin of healthy human participants [48].When applied as a nanocrystalline silver dressing for four to six days, AgNPs penetrated beyond the stratum corneum and reached the reticular dermis.Besides, an oral dosage of 10 ppm of AgNPs (5-10 nm) produced no physical metabolic, hematologic, urine, or imaging morphology changes in humans [50].

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Te Scientifc World Journal Although the included studies support the antibacterial efects of AgNPs against intracanal pathogens like E. faecalis, most of the results were derived from in vitro investigations.Te antibacterial properties of AgNPs depend on their type, formation method applied, and concentration.Moreover, their low dose was efective as an intracanal medicament, specifcally E. faecalis.However, caution should be exercised as signifcant usage of AgNPs in sublethal concentrations may lead to bacterial resistance, which was not evaluated in the included studies.Further studies are necessary to evaluate the ideal concentration of AgNPs for favorable antimicrobial efects without inducing cytotoxicity in vivo.As AgNPs produce synergistic antibacterial efects when combined with commonly used medicaments such as Ca(OH) 2 and CHX, newer NP-based formulations should be produced for successful root canal therapy.Besides, their tooth discoloration potential should be investigated.

Other Applications of AgNPs
AgNPs have various medicinal uses such as antimicrobials, antiviral, nematicidal, anthelmintic, anticancer, bone healing, wound repair promoters, vaccine adjuvants, antidiabetic agents, and biosensors as explained in an elaborate review of AgNPs by Xu et al. [51].

Antimicrobial, Antiviral, Nematicidal, and Anthelmintic
Activity.AgNPs inhibit pathogenic bacteria, fungi, and viruses, including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, dermatophyte, and HIV-1.As they are broad-spectrum antibacterials, they are applied in catheter modifcation, wound healing, and bone healing [51].In addition, are antiviral against hepatitis B virus (HBV) [52], human parainfuenza virus (HPIV), herpes simplex virus (HSV) [53], and infuenza A (H1N1) virus [54].AgNPs are helpful antivirals due to their reduced size (<10 nm), which enhances their large surface area and ability to adhere to the virus surface.Tey bind to the glycoprotein knobs, inhibit the reverse transcriptase enzyme, and interact with the virus in size-and dose-dependent manner [55].Tey either block the virus's contact with cells or directly inactivate it.Furthermore, they alter the cellular targets responsible for drug resistance and pathogenicity in resistant fungi.In C. albicans, AgNPs act on oleic acid targets that are signifcant for hyphal morphogenesis and pathogenicity.Tey saturate, adhere to the fungal hypha, and inactivate the fungus [51,56].

Anticancer Efect.
AgNPs produce a wide range of anticancer efects as they infuence cancer cells' proliferation, viability, apoptosis, and necrosis by destroying their ultrastructure, inducing ROS production, and DNA damage [51].AgNPs promote apoptosis by regulating the expression of vital genes like p53 that regulate essential signaling pathways, such as the hypoxia-inducible factor pathway.It induces sub-G1 cell cycle arrest and apoptosis in several cancer cells exposed to AgNPs [51,60].Tey also prevent metastasis by impeding tumor cell migration and angiogenesis [51,61].

Wound Repair and Bone
Healing.AgNPs increase the wound healing rate and produce notable cosmetic results with near-average hair growth and reduced hypertrophic scarring.AgNPs reduce TGF-β levels, increase interferon-c and vascular endothelial growth factor mRNA in keratinocytes at the wound edge, and promote wound healing by inducing angiogenesis [51].Tey persist in the fbroblast cytoplasm and stimulate dermis and epidermis restoration.Tey induce the proliferation and migration of keratinocytes, reduce collagen and hydroxyproline levels, and promote fbroblast diferentiation into myofbroblasts, leading to early wound adhesion, contraction, and closure [51,62].

Vaccine Adjuvant.
Te vaccine adjuvants reduce the amount of antigen required, shorten the time required for a protective threshold of antibody production, improve the intensity of the elicited responses, and stimulate long-term memory responses to reduce the requirement of repeated vaccinations.AgNPs are immunological adjuvants as they stimulate T2-biased immune responses through increased serum antigen-specifc IgG and IgE production and activate and recruit local leukocytes and macrophages [51].

Antidiabetic Agent.
AgNPs derived from plant extracts are antidiabetic.For example, AgNPs synthesized from the Solanum nigrum leaf extract reduced the blood glucose levels in diabetic rats [51,64].Compared to glibenclamide, they produced an excellent hypoglycemic efect.Similarly, AgNPs synthesized from the Argyreia nervosa leaf extract inhibited α-amylase and α-glucosidase, producing an antidiabetic efect [51,65].AgNPs infuence insulin signaling or sensitivity by activating protein kinase C and PI3K pathways at the insulin receptor substrate level.Tey inhibit protein kinase C isozyme, enhance insulin sensitivity and secretion, and reduce insulin resistance [51].
5.6.Biosensor and Imaging.AgNPs are a cost-efective surface-enhanced Raman scattering substrate (SERS) [51].Tey are applied as biosensors to identify blood glucose, enzymes, tumor markers, and pathogens.Te nanostructure and large surface area of carriers enhance the interaction between AgNPs and electrodes and accelerate the electron transfer of AgNPs, leading to improved biosensor sensitivity Te Scientifc World Journal [51,66].For instance, AgNPs combined with graphene oxide NPs enabled SERS biosensing and drug delivery [51,67].Tey are also used as synthetic probes to detect mercury content in water, soil, and food, detect copper ions in blood samples, and detect sickle cell anemia mutations [51].

Conclusion
Tis review shows that AgNPs have antibacterial efcacy comparable to Ca(OH) 2 , and this efect increases when the two materials are combined and used as intracanal medicaments.However, further in vivo clinical trials are needed to determine the efective AgNP antibacterial concentrations and plausible adverse efects.

Table 1 :
Evidence search for the role of AgNPs as an intracanal medicament.Risk of bias assessment of in vitro studies and randomized controlled clinical trials.

Table 2 :
Studies reporting antimicrobial efect of AgNPs as an intracanal medicament.
ix) Efective antibacterial activity of AgNPs against E. faecalis bioflm [30] Combination of AgNPs with Ca(OH) 2 was more efective against F. nucleatum Fahim et al.[30](xvi) Antibacterial efect of the AgNPs and nano-Ca(OH) 2 was equivalent to that of Ca(OH) 2 but produced better pain control